The objective of this R21 research proposal is to develop an instrument that can reliably manipulate small amounts of sample to rapidly and automatically perform hydrogen/deuterium exchange (HDX) and be interfaced with current HPLC-ESI-MS systems. We envision this device to be a versatile appendage module to any ESI-MS, already found in many proteomic, biology, and pathology laboratories, and to enable them to perform HDX experiments with minimal human operation. Our long-term goal is to make HDX technology more accessible to a far greater number of researchers who can utilize it to explore protein conformations and dynamics in their studies, at a sensitivity and throughput unmatched by any current instrumentation. Mass spectrometry-based HDX (HDX-MS) experiments probe protein structures by monitoring the rate and extent of deuterium exchange with backbone amide protons. This approach has proven to be a powerful and versatile protein analysis technique, which can provide valuable insights into protein dynamics in solution such as protein folding, protein-protein complex formation and protein-ligand interactions. Despite these attractive features of HDX technology and its many useful applications, the extensive sample handling necessary to produce the labeled protein provides in itself a source of error, particularly with short incubation times and manual pipetting. Therefore, automation of the labeling procedure would be an advantage. We have developed an automated digital microfluidic droplet generator (DMDG) platform that can generate nanoliter droplets with precisely defined compositions pre-programmed by the user. Our joint team has demonstrated automated multi-step HDX experiments with two model proteins, myoglobin (Mb) and bacteriorhodopsin (bR, an integral membrane protein) using the first-generation DMDG chips coupled with intact protein MS analysis. Herein, we will develop a DMDG microfluidics instrument, toward minimizing the amount of sample needed, and maximizing the speed of output, under the precise experimental conditions required for HDX experiments. We propose two different chips with (1) parallel channel and (2) variable-volume, single channel designs for HDX testing. Subsequently, we will validate the performance of the second-generation DMDG platform with superoxide-dismutase (SOD1) by site-specific HDX using (1) rapid electron capture/transfer dissociation (ECD/ETD) of intact proteins and (2) pepsin column digestion for peptide validation. Finally, we will explore the possibility of high-throughput HDX-MS using the optimal DMDG chips and multiple micro-size-exclusion chromatography or reverse-phase trap systems. The particularly useful HDX-MS methods, along with the established basic science and engineering programs of our labs, make our mass spectrometry and microfluidic team (J. Whitelegge and C. K.-F. Shen) uniquely positioned to translate new technologies into ongoing scientific discovery at UCLA.